Deacetylation via SIRT2 prevents keratin-mutation-associated injury and keratin aggregation

Keratin (K) and other intermediate filament (IF) protein mutations at conserved arginines disrupt keratin filaments into aggregates and cause human epidermolysis bullosa simplex (EBS; K14-R125C) or predispose to mouse liver injury (K18-R90C). The challenge for more than 70 IF-associated diseases is the lack of clinically utilized IF-targeted therapies. We used high-throughput drug screening to identify compounds that normalized mutation-triggered keratin filament disruption. Parthenolide, a plant sesquiterpene lactone, dramatically reversed keratin filament disruption and protected cells and mice expressing K18-R90C from apoptosis. K18-R90C became hyperacetylated compared with K18-WT and treatment with parthenolide normalized K18 acetylation. Parthenolide upregulated the NAD-dependent SIRT2, and increased SIRT2-keratin association. SIRT2 knockdown or pharmacologic inhibition blocked the parthenolide effect, while site-specific Lys-to-Arg mutation of keratin acetylation sites normalized K18-R90C filaments. Treatment of K18-R90C–expressing cells and mice with nicotinamide mononucleotide had a parthenolide-like protective effect. In 2 human K18 variants that associate with human fatal drug-induced liver injury, parthenolide protected K18-D89H– but not K8-K393R–induced filament disruption and cell death. Importantly, parthenolide normalized K14-R125C–mediated filament disruption in keratinocytes and inhibited dispase-triggered keratinocyte sheet fragmentation and Fas-mediated apoptosis. Therefore, keratin acetylation may provide a novel therapeutic target for some keratin-associated diseases.


Introduction
Intermediate filaments (IFs) make up a large family of cytoplasmic and nuclear cytoskeletal proteins that are expressed in a cell-selective manner and whose mutation accounts for more than 70 IF-associated diseases (IF-pathies) (1)(2)(3)(4)(5). Among IFs, keratins make up the largest family, with 54 functional keratin genes (6). Keratins include type I (K9-K28, K31-K40) and type II (K1-K8, K71-K86) proteins, which are expressed as obligate noncovalent heteropolymer pairs in an epithelial cell-specific manner (7) (e.g., K8/ K18 in simple-type epithelia, K5/K14 in basal keratinocytes). A common structural feature among all IFs is a conserved central α-helical rod domain that is flanked by less conserved N-terminal head and C-terminal tail domains (1,7). Among IFs, R90 of K18 is a highly conserved residue, and its mutation in epidermal keratins (K14-R125C) was the first link of an IF mutation to any human disease (in this case, epidermolysis bullosa simplex [EBS]; see refs. 8,9). The K18-R90C mutation, when expressed in cultured cells or as a transgene in mice, causes the collapse of liver keratin filaments into dots and aggregates, and leads to keratin hyperphosphorylation and predisposition to Fas-induced and several other types of liver injury (10)(11)(12). Similarly, mutations at this highly conserved residue and other conserved residues (e.g., K18-D89H and Keratin (K) and other intermediate filament (IF) protein mutations at conserved arginines disrupt keratin filaments into aggregates and cause human epidermolysis bullosa simplex (EBS; K14-R125C) or predispose to mouse liver injury (K18-R90C). The challenge for more than 70 IF-associated diseases is the lack of clinically utilized IF-targeted therapies. We used highthroughput drug screening to identify compounds that normalized mutation-triggered keratin filament disruption. Parthenolide, a plant sesquiterpene lactone, dramatically reversed keratin filament disruption and protected cells and mice expressing K18-R90C from apoptosis. K18-R90C became hyperacetylated compared with K18-WT and treatment with parthenolide normalized K18 acetylation. Parthenolide upregulated the NAD-dependent SIRT2, and increased SIRT2-keratin association. SIRT2 knockdown or pharmacologic inhibition blocked the parthenolide effect, while site-specific Lys-to-Arg mutation of keratin acetylation sites normalized K18-R90C filaments. Treatment of K18-R90C-expressing cells and mice with nicotinamide mononucleotide had a parthenolide-like protective effect. In 2 human K18 variants that associate with human fatal drug-induced liver injury, parthenolide protected K18-D89H-but not K8-K393R-induced filament disruption and cell death. Importantly, parthenolide normalized K14-R125C-mediated filament disruption in keratinocytes and inhibited dispase-triggered keratinocyte sheet fragmentation and Fas-mediated apoptosis. Therefore, keratin acetylation may provide a novel therapeutic target for some keratin-associated diseases.
Given the lack of directed therapy toward the diverse IF-associated diseases, we undertook an unbiased high-throughput drug-screening approach using A549 human lung adenocarcinoma cells that were transduced with green fluorescent protein (GFP)-K18-R90C. High-throughput drug screening has been utilized to identify compounds that stabilized IFs and potentially serve as direct or indirect chemical chaperones (16). This approach has successfully identified the pan-kinase inhibitor PKC412 as a compound that reverts disrupted K18-R90C aggregates into a WT-like extended filament network by enhancing keratin association with nonmuscle myosin heavy chain IIA (NM-IIA) in a myosin-dephosphorylation-regulated manner (17), or in patient-derived keratinocytes expressing K14-R125C (18). Also, by using this high-throughput drug screening system, another compound termed PP2, a SRC-family tyrosine kinase inhibitor, was identified and demonstrated to protect cells from keratin-mutation-associated liver injury and filament disruption via SRC kinase inhibition (19). Our hypothesis is that compounds that convert the mutant keratin phenotype from dots/aggregates to filaments will protect cells and tissues from injury. The power of this approach is that a reverse mechanistic approach can be utilized to identify keratin regulatory mechanisms.
Posttranslational modifications (PTMs) play an important role in regulating the assembly and disassembly dynamics of IF proteins, as well as their associations with other cellular components (20)(21)(22). Several stress-induced PTMs, including phosphorylation, transamidation, and sumoylation, have been well studied (23)(24)(25). Another keratin regulatory modification is acetylation, as exemplified by acetylation at the K8 conserved residue Lys207, which regulates keratin solubility and filament organization (26). Several other K8/K18 acetylation sites have been identified using high-resolution mass spectrometry (27,28), but the function of IF acetylation and its crosstalk with other IF PTMs remain poorly understood (22).
In the present study, we used high-throughput dug screening to identify the plant derivative parthenolide (PN) as a compound that potently normalizes K18-R90C-mediated keratin filament aggregation in cells and mouse liver, which in turn protected cells and mice from Fas ligand-induced (Fas-L-induced) apoptosis. Moreover, we demonstrate that PN treatment results in deacetylation of K8/K18 via increased binding with NAD-dependent sirtuin 2 (SIRT2). The importance of keratin deacetylation was supported by pharmacologic inhibition or molecular knockdown of SIRT2, site-specific mutation of keratin acetylation sites in the context of K18-R90C, and treatment of K18-R90C-expressing cells and mice with nicotinamide mononucleotide (NMN). Importantly, the effectiveness of PN extended to the natural variants K18-D89H and K14-R125C. Our findings provide multiple lines of evidence that keratin acetylation provides a potential therapeutic target for some of the IF-pathies by normalizing keratin-mutation-induced filament disorganization.

Results
PN treatment normalizes K18-R90C filament disruption and protects against Fas-induced apoptosis in cultured cells and mice. We used lentivirally transduced A549 cells that express GFP-K18-R90C as a system to screen for compounds that normalize mutation-induced keratin filament disruption. Compounds of interest were those that decreased the number of keratin dots (i.e., keratin aggregation) per cell, as assessed by fluorescence imaging. Based on a screening of a 1,037-compound library, PN was selected for further characterization since it is a well-studied natural product (29). After treating K18-R90C cells with PN or DMSO as control vehicle, PN had a dramatic effect in normalizing the disrupted K18-containing filaments by decreasing the percentage of cells with dots from 65% to 30% ( Figure 1A and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.166314DS1), without altering K8/K18 or other keratin levels (Supplemental Figure 1B). Compared with PKC412, which also reverts disrupted K18-R90C filaments into WT-like extended filaments by enhancing keratin-myosin association in a myosin-dephosphorylation-regulated fashion (17), PN showed earlier significant effects (Supplemental Figure 1, A and C). We also tested dimethylaminoparthenolide (DMAPT), a soluble analog of PN on K18-R90C cells. DMAPT showed a similar corrective effect to that of PN on R90C-induced filament disorganization (Supplemental Figure 1D). One of the major potential benefits of identifying keratin filament-normalizing drugs is to find compounds that can overcome the R90C-induced predisposition to cell injury. Importantly, PN protected A549 cells that express K18-R90C but not K18-WT from apoptosis (induced by IFN-γ and Fas-L), as determined by the decreased level of the apoptosis markers, cleaved PARP and caspases 3 and 7 ( Figure 1B). TUNEL assay showed markedly less positive nuclear staining after PN treatment ( Figure 1C and Supplemental Figure 1E), further supporting the conclusion that PN protects from apoptosis in K18-R90C-expressing cells. We then tested the effectiveness of PN in vivo. Administration of PN to transgenic mice that express human K18-R90C led to keratin filament normalization in the livers, with decreased abnormal dot-pattern keratin staining from 74% to 32% ( Figure 1D and Supplemental Figure 1F), without altering K8/ K18 levels (Supplemental Figure 1G). Similar results were seen in livers of the mutant mice fed DMAPT by gavage (Supplemental Figure 1H). Importantly, PN protected K18-R90C-expressing mice from Fas-L-induced liver injury, as evidenced by significantly decreased apoptosis markers in liver lysates ( Figure 1E), lower serum ALT levels ( Figure 1F PN decreases K18 acetylation via SIRT2 in cells and mice. We explored the mechanism underlying PN's effect. Previously, PN was reported to promote HDAC1 depletion and cell death (29), and to lead to regression of cholestasis-induced fibrosis by inhibiting HDAC4 (30). Given our prior characterization of K8 acetylation at Lys-207 and its role in decreasing keratin solubility (26), we examined the acetylation of K18 by performing anti-AcK immunoprecipitation followed by blotting for bound keratins. Notably, K18-R90C was highly acetylated compared with K18-WT, and PN treatment led to a prominent decrease in K18-R90C acetylation ( Figure 2A). This deacetylation was supported by carrying out the reciprocal experiment (i.e., anti-GFP immunoprecipitation followed by anti-Ack blot; Figure 2B). We then tested whether the major cytoplasmic deacetylases, SIRT2 and HDAC6, are involved in K18-R90C deacetylation.
Transfection of FLAG-tagged SIRT2 or HDAC6 in BHK or A549 cells showed that SIRT2, but not HDAC6, decreased K18 acetylation (Supplemental Figure 2A). Furthermore, K18-R90C-expressing cells had lower levels of SIRT2 (compared with WT-expressing cells) that increased after PN treatment, commensurate with a decrease in SIRT2 substrate (α-tubulin) acetylation ( Figure 2C). To explore whether PN could regulate other sirtuin family members, we tested the mRNA levels of SIRT1-SIRT7 in WT and R90C cells upon PN treatment. The results revealed that PN upregulated the mRNA levels of SIRT2 and SIRT5 (Supplemental Figure 2, B-H). However, PN did not alter the protein level of SIRT5 (Supplemental Figure  2I). Thus, we focused on SIRT2 in all subsequent experiments. To test whether PN can activate SIRT2 directly, we performed an in vitro fluorescence-based assay, which showed that PN increases SIRT2 activity at concentrations of 5 μM or higher ( Figure 2D). Moreover, keratin-SIRT2 colocalization increased after PN treatment of K18-R90C cells, as determined by immunofluorescent staining and coimmunoprecipitation ( Figure 2, E and F, and Supplemental Figure 2J).
We then assessed the distribution of SIRT2 and K18 in K18-R90C livers in the presence or absence of PN, by sequential fractionation with a detergent-free buffer, followed by nonionic (NP-40) and zwitterionic (Empigen) detergent solubilization and compared these fractions to the remaining pellet. As shown in Supplemental Figure 3A, SIRT2 and K18 become more soluble upon cell exposure to PN (e.g., K18 moves from the pellet to the Empigen fraction after PN treatment, and SIRT2 levels decrease in the pellet fraction and are more readily detectable in the hypotonic fraction). In addition, K18 and α-tubulin acetylation in livers of K18-R90C mice was downregulated after 4 daily injections of PN ( Figure 2G and Supplemental Figure 3B). cells were transduced with lentivirus expressing GFP-tagged K18-R90C and K8-WT (cotransfection with type I and II keratins is needed for physiologic heteropolymer filament formation). After 2 days, cells were treated with DMSO or PN (5 μM, 48 hours) and then fixed and stained with DAPI. Average percentage of cells with dots/group for DMSO vs. PN is shown. P < 0.001, from 3 independent experiments). Scale bar: 50 μm. (B) GFP-K18-WT and GFP-K18-R90C lentiviruses were transduced into A549 cells followed by treatment with DMSO or PN (5 μM, 48 hours) and then apoptosis was induced using IFN-γ and Fas ligand (Fas-L). Cell lysates were analyzed by blotting using antibodies against the indicated antigens (Coomassie stain shows equal loading). The average relative intensity of the indicated bands from 3 individual experiments is included below each blot. *P < 0.05, ***P < 0.001 using unpaired, 2-tailed Student's t test (comparing DMSO with PN). (C) Representative TUNEL staining of GFP-K18-R90C-transduced A549 cells treated with DMSO or PN and then challenged with IFN-γ plus Fas-L. The percentage of TUNEL + cells in DMSO vs. PN was 82% vs. 33%, respectively (P < 0.001). Scale bar: 50 μm. For panels A-C, all experiments were repeated at least 3 times. *P < 0.05, **P < 0.005 using unpaired, 2-tailed Student's t test. (D) Transgenic mice that express K18-R90C were treated daily with DMSO or PN (1 mg/kg mouse weight; i.p.) for 4 days. Liver sections were double stained with anti-K18 antibody and DAPI. The percentage of cells with dots/group is shown in Supplemental Figure 1F  Moreover, the association between human K18 and SIRT2 increased prominently, as demonstrated by SIRT2 immunoprecipitation followed by blotting for K18 ( Figure 2H).
Inhibition of SIRT2 impairs the ability of PN to ameliorate K18-R90C aggregation. The importance of SIRT2-mediated deacetylation of K18 in normalizing mutant keratin filament disruption was further supported by treating K18-R90C cells with the SIRT2-selective inhibitor AGK2 in the presence or absence of PN. The GFP-K18 fluorescence showed that AGK2 blocked PN's effect of correcting the R90C-induced filament disruption and increased K18 acetylation ( Figure 3A and Supplemental Figure  4A). Similarly, AGK2 treatment inhibited PN's activation of SIRT2, as determined by α-tubulin acetylation ( Figure 3B). Similar findings regarding keratin acetylation and filament organization were observed using siRNA-mediated SIRT2 knockdown (Figure 3, C and D, and Supplemental Figure 4B). We also found that SIRT5 knockdown did not impair PN's effect on K18-R90C-induced filament disorganization and K18 deacetylation (Supplemental Figure 4, C and D, respectively).
NMN normalizes K18-R90C-induced keratin filament disruption. In order to further support the importance of (the NAD-dependent, ref. 31) SIRT2 in keratin filament normalization, we examined the effect of NMN in cells and mice that express K18-R90C. There are 4 pathways to synthesize NAD+, including the salvage pathway that utilizes nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR), or the de novo pathway that uses tryptophan (32). Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ biosynthetic pathway, produces NMN in 1 step, a key NAD+ intermediate. In many studies, administration of NMN enhances NAD+ biosynthesis in vitro and in vivo, thereby rendering NMN a key precursor with possible therapeutic implications (33)(34)(35)(36). This led us to test the effect of NMN on K18-R90C-mediated keratin filament disruption in A549 cells. Strikingly, NMN ameliorated mutant keratin filament aggregation commensurate with decreased K18 acetylation and protected the cells from Fas-induced apoptosis without having an additive effect with PN ( Figure 4, A-C). In vivo, NMN improved liver keratin filament organization, together with decreased K18 acetylation and, importantly, protected the mice from Fas-induced liver injury (Figure 4, D-G).

. Keratin deacetylation by nicotinamide mononucleotide (NMN) normalizes K18-R90C-induced keratin filament disruption and protects against Fas-induced apoptosis in cells and mouse liver.
(A) A549 cells were transduced with GFP-K18-R90C followed by treatment with NMN (2 mM), PN (5 μM) or NMN + PN. Cells were imaged after counterstaining the nuclei (blue). Scale bar: 50 μm. Quantification of the percentage cells with dots was done using pooled counts from 3 experiments. Data are presented as mean ± SD. *P = 0.03, **P < 0.01, ***P < 0.001 using 1-way ANOVA followed by Tukey's post hoc test. (B) Lysates from cells similar to those used in panel A were used for immunoprecipitation with an anti-AcK antibody followed by immunoblotting with an anti-K18 antibody (the input lysate was similarly blotted). (C) Cells as in panel A were treated with IFN-γ and Fas-L to induce apoptosis. Cell lysates were analyzed by blotting using antibodies against the indicated antigens. (D) Mice that overexpress K18-R90C were treated daily with PBS or NMN for 4 days. Liver sections were then double stained with anti-K18 antibody (red) and DAPI (blue). P = 0.01 when comparing the percentage cells with dots in hepatocytes from PBS vs. NMN treatment groups. (E) Livers from 2 pairs of K18-R90C mouse siblings were treated with NMN or PBS (4 days) followed by solubilization, AcK immunoprecipitation, and then blotting (of precipitates and input) with an anti-K18 antibody. (F) Mice were challenged with Fas-L to induce liver injury, followed by measurement of serum alanine transaminase (ALT). *P < 0.03 using an unpaired, 2-tailed Student's t test. (G) Livers from mice used in panel E were homogenized followed by blotting with antibodies against the indicated antigens. HSP70 is included as a loading control. Each lane represents livers from separate animals. of mutating K18 acetylation (in the background of also having K18-R90C), is not surprising due to the obligate heteropolymeric organization of keratins and the human disease phenotype whereby mutation of type I or type II keratins typically behaves similarly (1,7,9,13,37).

PN normalizes K14-R125C-and K18-D89H-induced filament disruption and protects from Fas-induced apoptosis.
We also tested the effect of PN in the context of the patient-associated mutations K14-R125C (which causes EBS), and K18-D89H, and K8-K393R (which associate with fatal isoniazid and ezetimibe/simvastatin-related drug-induced liver injury, respectively; ref. 15). We transduced YFP-K14-WT or K14-R125C in keratinocytes deficient in type I keratins (38), followed by treatment with PN or carrier. Importantly, PN cleared the keratinocyte punctate staining significantly ( Figure 6A). Moreover, PN increased cell adhesion and resistance to mechanical stress, as determined using a dispase assay (39). As such, an intact epithelial sheet of K14-WT cells lifted off the culture dish in response to mechanical force, while K14-R125C-expressing cells displayed multiple cell-sheet fragments, with PN leading to rescue of the sheet fragmentation ( Figure 6B). Furthermore, PN protected K14-R125C cells from IFN-γand Fas-L-induced apoptosis, as determined by decreased TUNEL staining and cleaved caspase-7 (Figure 6, C and D). Similarly, PN protected CHO cells transduced with K18-D89H from isoniazid-induced cell apoptosis (Figure 7). The PN protective effect appears to be keratin mutant site-specific since it did not protect cells expressing the K8-K393R variant (Supplemental Figure 6). Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 when comparing each of the double mutant constructs with cells transfected with WT K8 and K18-R90C. One-way ANOVA was used for comparison between treatment groups, and Tukey's post hoc test was used for comparing multiple groups with the R90C/WT group.

Discussion
In the present study, an unbiased drug screening approach led us to identify PN as a compound with a profound normalizing effect on mutation-triggered keratin filament disruption and consequent protection from apoptosis and injury in cell culture systems and in mice (Figure 1). This finding led us, unexpectedly, to a link between keratin filament organization and the acetylation pathway whereby we demonstrate that K18-R90C mutation leads to keratin hyperacetylation, which is inhibited by PN (Figures 2 and 3). Of note, PN did not protect against apoptosis in a WT keratin background ( Figure 1B and Supplemental Figure 1, I-K). We attribute this difference to the selective effect of PN on the hyperacetylated keratin mutant (Figure 2A), which implies that the filament-disrupting keratin mutation is necessary for PN to manifest its protective effect. The importance of keratin deacetylation in normalizing keratin filament was then conclusively demonstrated using pharmacologic means, knockdown of SIRT2, and site-selective mutation of K18 or K8 acetylation in the context of K18-R90C (Figures 2-5 and Supplemental Figures 4 and 5). The importance of sirtuins was further validated by showing that the sirtuin activator NMN has a PN-like effect in terms of keratin filament Quantification of the percentage cells with dots/group using pooled counts from 3 separate experiments is shown in the histogram on the right (*P = 0.013, ***P < 0.001). Imaging of the YFP-tagged K14 was done by converting the yellow YFP to a green pseudo color (i.e., from the yellow [570-590 nm] to the green [495-570 nm] wavelength). (B) Cells as in panel A were treated with dispase to test resistance of the epithelial sheet to mechanical stress. Fragments of the cell sheet were counted and quantified (***P < 0.001). (C) Representative images of TUNEL-stained keratinocytes transduced with K14-R125C and then treated with DMSO or PN (5 μM, 48 hours) followed by IFN-γ + Fas-L to induce apoptosis. Quantification of the percentage TUNEL + cells using pooled counts from 3 separate experiments is shown on the right. ****P < 0.001 using unpaired, 2-tailed Student's t test. Scale bar: 50 μm. (D) Lysates of cells, similar to those used in panel C, were blotted with antibodies against caspase-7 or cleaved caspase-7. For panels A and B, 1-way ANOVA was used for comparison between treatment groups, and Tukey's post hoc test was used for 2-group comparisons.  (Figure 4). The effect of PN on K18-R90C extends to the K18-D89H variant that is associated with fatal isoniazid-induced liver injury, and to the epidermal K14-R125C mutation that causes the blistering disease EBS (Figures 6 and 7).
Similarly protective effects of PN in liver were observed in other contexts. For example, Cui et al. reported that PN ameliorates fibrogenesis and inflammation in hepatic fibrosis (40). Also, PN exerts beneficial effects on liver injury, lipid metabolism, fibrosis, inflammation, and oxidative stress in mice with metabolic dysfunction-associated fatty liver disease (41). Moreover, PN exhibited protective effects in rodent models of nonalcoholic fatty liver disease and concanavalin A-induced acute hepatitis (42,43). Although the underlying mechanisms of the previously observed hepatoprotective effects of PN varied in different conditions, most of them were related to the antiinflammatory function of PN. In our study herein, we uncovered that PN protects from genetic keratin-mutation-triggered liver injury by modifying the acetylation of K8/K18, with consequent normalization of keratin filament organization. Although PN appears to have several biologic effects, we link its effect on acetylation, as supported by the findings of SIRT2 knockdown and pharmacologic intervention.
Among all IF posttranslational modifications, phosphorylation is the best studied, with keratin hyperphosphorylation being a hallmark that accompanies essentially every form of epithelial cell stress (22). In that context, the multikinase inhibitor PKC412 had a similar effect to that of PN in normalizing K18-R90C dots (17), although PN manifested an earlier beneficial effect (Supplemental Figure  1C). However, enzyme activators typically offer several advantages as compared with inhibitors, such as having greater target specificity (44). Also, while some double K8 but not K18 phospho (Ser-to-Ala)/ R90C mutants led to conversion of keratin dots to filaments, triple acetyl-phospho-R90C mutation of K8/K18 had no added benefit in normalizing keratin filaments, and some phospho/combined mutants resulted in more dots (Supplemental Figure 7). This suggests that keratin deacetylation may be upstream of phosphorylation, or that keratin deacetylation may have a more dominant effect compared with hypophosphorylation. More importantly, PKC412 acts by dephosphorylating myosin rather than major K8/ K18 phosphorylation sites (17), and PKC412 does not lead to K18 deacetylation (not shown).
PN has also been studied as an NF-κB inhibitor (45). PN was characterized as an NF-κB inhibitor that can directly inhibit the p65 subunit and has been used as an NF-κB inhibitor in several studies (46,47), aside from other finding describing its role in acetylation (28,29). Of note, SIRT2 interacts with NF-κB cytoplasmic p65 and deacetylates p65 at Lys-310, leading to its inactivation (48). Our findings herein provide another link between PN and SIRT2.
The role of sirtuin pathways and acetylation has been reported previously in hepatic pathophysiology. For example, hepatic SIRT2 protein expression was inversely correlated with alcoholic liver disease development (49). Moreover, SIRT1-mediated deacetylation regulates hepatic lipid metabolism and controls the regenerative response of liver (50,51). In our study herein, the link of deacetylation to mutant keratin filament normalization was based on several lines of evidence, including point mutations of keratin acetylation, pharmacologic inhibition and activation of SIRT2, and knockdown of SIRT2. We examined keratin acetylation using an anti-acetylated lysine antibody, but the 3 antibodies we tested from different vendors show varied patterns (Supplemental Figure 8), which suggested that these antibodies have different acetylation site recognition patterns. For example, for some of the antibodies, PN in some cases led to decreased antibody reactivity (deacetylation), while other proteins appeared to undergo hyperacetylation after PN treatment. This indicates that PN's effect on acetylation varies, depending on the protein. These findings also suggest that PN may not be a pan-inhibitor or pan-activator of acetylation, which explains the seemingly different PN inhibitory effect on HDAC1 and -4 (29, 30) but activation of SIRT2.
The insolubility of PN limits its clinical utility, but its water-soluble prodrug DMAPT has PN-like biologic effects and might prove to be useful clinically (52). Our findings showed that DMAPT has similar normalizing effects on mutant keratin filament disorganization in cell culture and in mouse liver as compared with PN (Supplemental Figure 1, D and H). PN is known to have pleiotropic effects, including antiinflammatory, antioxidative, and antifibrotic effects, as well as linkage with multiple signaling pathways, including NF-κB, p53, HIPPO, TLR4, and STAT3 (40)(41)(42)(43)45). PN also destabilizes microtubules by forming tubulin adducts on cysteine and histidine residues (53). We hypothesize that compounds targeting SIRT2 directly (e.g., NMN) are more likely to be clinically applicable for the keratin mutations we studied, and offer an exciting potential particularly given that several sirtuin-activating compounds are presently undergoing clinical trials (54).
In conclusion, the targeting of simple-epithelial K18 and epidermal K14 acetylation, in the context of K18 and K14 mutations that have dramatic clinical consequences (fatal drug-induced liver injury/blistering skin disease) and cell biologic manifestations (morphologic change from filaments to dots with the biologic consequence of IF aggregation and predisposition to apoptosis), offers potential therapeutic options and a mechanism for regulating keratin filament organization (Figure 8). One potential benefit of identifying uniquely functioning drugs that may have clinical utility in keratin-and other IF-related disorders is that they may offer the opportunity to be used, after testing experimentally, as combination drugs. This applies to the demonstrated protective effect in keratin mutants by 2 separate Ser/Thr or Tyr kinase inhibitors (17)(18)(19) and the observation herein by targeting protein acetylation. Such an approach is likely to be particularly beneficial for drugs that are already in clinical use and, therefore, can be repurposed for keratin-and other IF-related disorders. Such drug combinations may offer several potential advantages, including decreased toxicity profiles (if effective at lower doses when combined) and having an additive or even synergistic effect.
Histology and immunofluorescent staining. Hematoxylin and eosin staining was performed by the Microscopy and Image Analysis Core at the University of Michigan. Frozen mouse liver sections or cells were prepared for immunofluorescent staining as described previously (55). Slides were fixed in acetone (10 minutes, -20°C), air dried for 20 minutes, and incubated in blocking solution (PBS, 2% bovine serum albumin, 2% goat serum). Primary antibodies (e.g., anti-keratin antibody L2A1, which recognizes human K18; ref. 12; see Supplemental Table 1 for a list of additional antibodies that were used in this study) were added (22°C, 1 hour) followed by 3 rinses with PBS and then addition of the secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Life Technologies). Staining was visualized using an Olympus FluoView-500 laser scanning confocal microscope and a 60x water-immersion (1.2 NA) objective and FluoView software (version 5.0; Olympus). For quantification, 10-20 fields were used for each treatment condition, and at least 3 individual experiments were repeated. Data are presented as mean ± SD of these individual experiments.
High-throughput screening. The screening strategy and procedure was as described in detail previously (16). Briefly, A549 cells (human lung carcinoma cell line, obtained from the American Type Culture Collection) were transduced with GFP-tagged K18-WT or K18-R90C, and K8-WT lentiviruses. This cell line was selected because it is nearly 100% transduced and it forms readily discernable extended filaments or dots, depending on the expression of WT or mutant keratins, respectively. Both K8 (type II keratin) and K18 (type I keratin) need to be present in order to form filaments, otherwise expression of a single type I or type II keratin in cells results in rapid degradation due to lack of stabilization by the partner keratin (56). After 1 day of transduction, cells were seeded into 384-well plates for screening, followed by addition of the compounds (Center for Chemical Genomics, University of Michigan) on day 2 after transduction. A total of 1,037 compounds were tested that include drugs that target the epigenetic, autophagic, redox, protease, kinase, Wnt, and cannabinoid pathways. After 2 days in the presence of the test compounds, cells were fixed for 20 minutes using 4% paraformaldehyde in PBS and then counterstained with 100 mg/mL 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) in PBS. Images were acquired using the Image Xpress Micro (IXM) XLS High Content Imaging System (Molecular Devices), followed by analysis using MetaXpress software (Molecular Devices). A total of 24 compounds were selected from the initial screening (performed in 384-well plates) for validation using chamber slides, with transduced A549 cells treated with the 24 compounds. From these compounds, PP2 (19) and PN had the most prominent effect in terms of decreasing the extent of K18-R90C filament collapse and dot formation. Further validation was then carried out by testing the dose response for PN (data not shown). For PN, the optimal dose for cell culture studies (in terms of maximal effect on normalizing the keratin filaments and having limited toxicity) was 5 μM. The stock solution of PN was 50 mM (dissolved in DMSO).
Cell culture, apoptosis induction, and construct transfection. CHO (Chinese hamster ovary), BHK (baby hamster kidney), and NIH/3T3 (mouse fibroblast) cells were obtained from the American Type Culture Collection and maintained as recommended by the supplier. NIH/3T3 cells were used when a system that lacks endogenous keratins was desired (they provide well-extended filaments). When sufficient protein expression was needed, BHK cells were used (which also lack keratins), although they do not provide the typical well-extended filaments (12,55). CHO cells were used for the K18-D89H and K8-K393R experiments since the typical keratin dot pattern with these constructs tended to revert to normal filaments in A549 cells after 3-4 days of transduction (data not shown). Mouse type I keratin-deficient keratinocytes were obtained from type I keratin-null embryos (38) followed by establishment of the cell line as described previously (57). To test susceptibility to apoptosis, GFP-K18-WT-and GFP-K18-R90C-transduced A549 cells were pretreated with DMSO or PN (5 mM, 48 hours) followed by addition of IFN-γ (R&D Systems; 40 ng/mL, 6 hours) and then Fas-L (CH11, Millipore; 100 ng/mL, 24 hours). Lipofectamine 2000 (Invitrogen) was used for